Effect of Protein Quality on Optimal Daily Protein Supply for Juvenile of Patin (Pangasianodon hypophthalmus)

EFFECT OF PROTEIN QUALITY ON OPTIMAL DAILY

PROTEIN SUPPLY FOR JUVENILE OF PATIN

(Pangasianodon hypophthalmus)

THESIS

DIAN RETNOSARI

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

DECLARATION

I do hereby declare that the thesis entitled “Effect of Protein Quality on

Optimal Daily Protein Supply for Juvenile of Patin (Pangasianodon

hypophthalmus)” is my original work produced through the guidance of my supervisors and that to the best of my knowledge it has not been presented for the award of any degree in any university. All of the incorporated material originated from other published as well unpublished papers and are stated clearly in the text as well as in the reference.

Bogor, May 2010

Dian Retnosari Reg.No. C151070261

ABSTRACT

DIAN RETNOSARI. Effect of Protein Quality on Optimal Daily Protein Supply for Juvenile of Patin (Pangasianodon hyphophthalmus). Under direction of DEDI JUSADI, and YANN MOREAU.

Two types of diet with different protein quality were formulated, namely High Fish Meal (HFM with 46.8 g CP kg-1) and Low Fish Meal (LFM with 46.3 g CP kg-1). Feeding rates were calculated to provide six different protein supplies 8, 14, 20, 23, 26, and 30 g kg-1 day-1 to duplicate groups of fish. Fishes were 3-inch length with an average mass 6.4±0.2 g. The density was 12 fish per aquarium. Daily ration was given in the 3 equal meals at 08.00, 12.00 and 16.00 for 6 weeks. Amino acid profiles show that HFM diet fulfilled requirement according to recommendations for channel catfish, while LFM diet appeared deficient in lysine. The specific growth rate, food conversion ratio, body fat, protein retention, fat retention and protein efficiency ratio were significantly different among treatments. On contrary, dry matter, protein, ash, gross energy did not differ significantly between treatments. Both dry matter and protein digestibility led to similar values for the two diets. Regarding response to protein supply, the results indicated that higher protein supply was required to reach maximum growth with LFM (17.8 g kg-1 day-1 for 2.04% day-1) than with HFM (16.1 g kg-1 day-1 for 2.5% day-1) diet. This suggests that increase in protein supply may partially compensate lower growth with lower dietary protein quality.

Keywords: stripped catfish, amino acid, protein quality, protein supply, Pangasianodon hypophthalmus.

iii SUMMARY

DIAN RETNOSARI. Effect of Protein Quality on Optimal Daily Protein Supply for Juvenile of Patin (Pangasianodon hyphophthalmus). Under direction of DEDI JUSADI, and YANN MOREAU.

Objective of this research was to study the effect of protein quality on optimal daily protein supply for juvenile of patin (Pangasianodon hypophthalmus). Previous studies concluded that the level and source of dietary protein significantly influenced the performance of channel catfish with supplemental amino acid such as crystalline lysine. While other studies suggested that amino acid in intact protein are more efficient than those provided in crystalline form because the latter are absorbed and catabolised more rapidly than the former ones. The present study aimed to provide information on dependence of growth to amino acid supply using a mixture of complementary proteins, in which different protein sources were mixed to modify the dietary amino acid profile.

This experiment used 3-inch patin juveniles with an average mass 6.4±0.2 g and density was 12 fish per aquarium. Two types of diet with different protein quality were formulated, namely High Fish Meal (HFM with 46.8 g CP kg-1) and Low Fish Meal (LFM with 46.3 g CP kg-1). Feeding rates were calculated to provide six different protein supplies 8, 14, 20, 23, 26, and 30 g kg-1 day-1 to duplicate groups of fish. The experimental period lasted for 6 weeks with weekly sampling to adjust daily ration. Daily ration was given in the 3 equal meals at 08.00, 12.00 and 16.00. Observed parameters were specific growth rate, survival rate, feed conversion ratio, protein retention, fat retention, and feed digestibility, as well as proximate body composition. Water quality (ammonia-N, pH, O2,

temperature and conductivity) was monitored weekly.

The survival of patin juveniles was 100%. The SGR, FCR, body fat, protein retention, fat retention and PER were significantly different among treatments. SGR increased with protein supply for both diet but reach a maximum at a lower level for HFM than for LFM diet. FCR were generally improved with increasing protein supply, but HFM diet gave a better result than LFM when protein supplies were equivalent. With the same protein supply, HFM diet gave a better result regarding protein retention. Fat retention decreased with increasing protein supply for both diet. Dry matter, protein, ash, GE did not differ significantly between treatments. Both dry matter and protein digestibility led to similar values for the two diets. Nevertheless, LFM diet led to slightly better value than HFM. Amino acid profiles of HFM diet fulfilled requirement given for channel catfish, even if excessive for most amino acids, while LFM diet appeared deficient in lysine, but fulfilled for the others. The results indicated that different in protein quality as resulting from difference amino acid supply led to different estimates of optimal protein supply. HFM diet which fulfilled amino acid requirement of patin body gave a better growth at high protein supply than LFM diet. Thus, protein supply with LFM diet must be increased to provide sufficient amount of lysine in order to allow full utilization of all amino acids. Then, it seems that higher protein supply may partially compensate unbalance amino-acid

iv ratio. In the present study, higher protein supply is effectively required for maximum growth with LFM diet.

Keywords: stripped catfish, amino acid, protein quality, protein supply, Pangasianodon hypophthalmus.

Copyright © 2010, by Bogor Agricultural University All rights reserved

1. No part or all of this thesis may be excerpted without inclusion or mentioning the sources

a. Excerption only for research and education use, writing for scientific papers, reporting, critical writing or reviewing of a problem

b. Excerption does not inflict a financial loss in the proper interest of Bogor Agricultural University

2. No part or all of this thesis may be transmitted and reproduced in any form without a written permission from Bogor Agricultural University

EFFECT OF PROTEIN QUALITY ON OPTIMAL DAILY

PROTEIN SUPPLY FOR JUVENILE OF PATIN

(Pangasianodon hypophthalmus)

DIAN RETNOSARI

Thesis

submitted in partial fulfillment of Master of Science degree in

Study Program of Aquaculture Science

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

Title of Thesis : Effect of Protein Quality on Optimal Daily Protein Supply for Juvenile of Patin (Pangasianodon hypophthalmus)

Name : Dian Retnosari Registration Number : C151070261

Approved

Advisors

Dr. Dedi Jusadi Dr. Yann Moreau

1st Advisor 2nd Advisor

Acknowledged

Head of Study Program Dean of Graduate School of Aquaculture Science

Prof. Dr. Enang Harris Prof. Dr. Ir. Khairil Anwar Notodiputro, M.S

ACKNOWLEDGEMENTS

I feel pleasure to express my sincere thanks and gratitude to my supervisor Dr. Dedi Jusadi and Dr. Yann Moreau for his relentless effort, guidance, supervision, encouragement and warm-hearted intelligence throughout my study at the Study Program of Aquaculture Science, Bogor Agricultural University. I do gratefully acknowledge the project and financial from the Institut de Recherche pour le Développement for this study. I wish to express my sincere appreciation and thanks to Rina Hirnawati, S.Pi, Nina Meilisza, M.Si and Ika Ayuningtyas, S.Si for their friendly help and co-operation with all analyses in the Nutrition Laboratories of IRD-BRBIH, Depok. Thanks are also due to Ruby Vidia Kusumah, S.Pi, Sawung Cindelaras, S.Pi, Rachmawati, S.Si and Irmayanti, S.Pi for their helpness along the experiment. My thanks are also extended to the technicians especially Subur. My heartfelt respect and special thanks must go to my beloved parents, brother, sister in law and all relatives back home whose sublime blessings, love and sacrifice inspired me most in my voyage to learning. Most especially I must thank to my friends in Aquaculture Science class of 2007 and lovely neighbour room, they are all my well-wishers. Finally, and by no means least important, I pledge my abiding gratitude to Deny Sapto Chondro Utomo, S.Pi for his helpness, patience, support and constant inspirations that undoubtedly helped me keep my sanity all through these hard times.

Bogor, May 2010 Dian Retnosari

AUTOBIOGRAPHY

The author was born in Tanjung Karang, June 19th 1984 from a father named Sarwadi, ST and a mother named Sriyani, A.Md. Author is a youngest child from two children.

In 2002 author was graduated from Bandar Lampung 2nd Senior High School and in the same time admitted to Padjadjaran University by National New Student Enrollment Selection. Author was chosen Fisheries and Marine Science Faculty and graduated with Fisheries Bachelor degree in 2007. In the same year, author was admitted to Graduate School, Bogor Agricultural University in Study Program of Aquaculture Science.

LIST OF CONTENT

Page

LIST OF TABLE ... xi

LIST OF FIGURE ... xii

LIST OF APPENDIX ... xiii

I. INTRODUCTION ... 1

1.1 Background ... 1

1.2 Objective ... 2

1.3 Hypothesis ... 2

II. LITERATURE REVIEW ... 3

2.1 Protein and Amino Acid ... 3

2.1.1 Fate of Protein ... 3

2.1.2 Amino Acid Rquirement ... 5

2.2 Patin (Pangasianodon hypophthalmus) ... 6

2.2.1 Classification and Morphology of Patin ... 6

2.2.2 Protein and Amino Acid Requirement ... 7

2.2.2.1 Protein Requirement ... 7

2.2.2.2 Amino Acid Requirement ... 8

2.3 Amino Acid Supply ... 9

III. MATERIALS AND METHODS ... 12

3.1 Rearing Condition ... 12

3.2 Diets ... 12

3.3 Chemical Analysis ... 12

3.4 Feed Digestibility Test ... 13

3.5 Experimental Method ... 13

3.6 Parameter ... 15

3.6.1 Specific Growth Rate ... 15

3.6.2 Survival Rate ... 15

3.6.3 Feed Conversion Ratio ... 15

3.6.4 Protein Retention ... 16

3.6.5 Fat Retention ... 16

3.6.6 Feed Digestibility ... 16

IV. RESULT AND DISCUSSION ... 17

4.1 Result ... 17

4.2 Discussion ... 22

V. CONCLUSION AND RECOMMENDATION ... 26

5.1 Conclusion ... 26

5.2 Recommendation ... 26

REFERENCE ... 27

xii

LIST OF TABLE

Page

1. Nutrient requirement of catfishes ... 8

2. Amino acid requirement of catfishes ... 9

3. Diets formulation ... 13

4. Experimental design with fixed protein supply ... 14

5. Average initial and final body mass, growth rate, and food conversion ratio according to diet and protein supply ... 19

6. Average of body composition according to diet and protein supply ... 20

7. Average of protein retention, fat retention, and protein efficiency ratio according to diet and protein supply ... 21

xiii

LIST OF FIGURE

Page

1. The fate of dietary protein ... 4 2. Patin (Pangasianodon hypophthalmus) ... 6 3. Dietary with good and bad protein content to satisfy

catfish requirement ... 10 4. Satisfying catfish requirement by increasing amount of

protein bound-lysine ... 11 5. Average mass gain (± SE) of patin fed on HFM diet during

experiment according to protein supply in g kg-1 day-1.

Each value corresponds to two observations ... 17 6. Average mass gain (± SE) of patin fed on LFM diet during

experiment according to protein supply in g kg-1 day-1.

Each value corresponds to two observations ... 18 7. Amino-acid profile in each feed compared with channel

catfish requirement (g kg-1 protein) ... 22 8. Broken line model adjusted to specific growth rate (SGR) of

patin juveniles according to daily protein supply with the two experimental diets over 33 days. Italic values indicated

minimum protein supply for maximal growth with each diet .. 23

xiv

LIST OF APPENDIX

Page

1. Proximate analysis of feed ingredients ... 34

2. Diet preparation procedure ... 35

3. Proximate analysis procedures ... 36

4. Random disposition of aquaria experiment ... 47

5. SGR and FCR calculation ... 48

6. Protein retention calculation ... 49

7. Fat retention calculation ... 50

8. Proximate analysis of patin body ... 51

9. ANOVA and Tukey test of SGR ... 52

10. ANOVA and Tukey test of FCR ... 53

11. ANOVA and Tukey test of PR ... 54

12. ANOVA and Tukey test of FR ... 55

13. ANOVA and Tukey test of PER ... 56

14. ANOVA and Tukey test of dry matter ... 57

15. ANOVA and Tukey test of protein ... 57

16. ANOVA and Tukey test of fat ... 58

17. ANOVA and Tukey test of ash ... 58

18. ANOVA and Tukey test of GE ... 59

19. Amino acid calculation ... 60

I. INTRODUCTION

1.1 Background

Patin (Pangasius sp.) is one of the eight pre-eminent species of aquaculture product that has been developed in Indonesia. This fish became a pre-eminent species when the technology for its culture has been controlled and disseminated to the public (DKP 2005). Expansion of patin production often faces constraint in culture management, feed, pest and disease. Therefore, efforts are needed to overcome these problems among them, a correct feeding either in quality and quantity. For the quality, fish requires feed with good nutritional value, easy to digest and easy to absorb.

Nutritional value of feed depends on its composition such as protein, carbohydrate, fat, vitamin, mineral and energy content. A fish feed must contain enough protein for growth and also for maintenance. Requirement of protein is different according to fish species, fish size, feedstuffs, protein quality and environment (NRC 1983). Its requirement will decrease along with size and age and it will increase along with temperature (Halver 2002). The availability of dietary protein can vary considerably among different feedstuffs. Fish meals are the primary source of protein in fish diet, including in the diet of channel catfish as they can provide all of the dietary essential amino acids required for growth, with a more favorable ratios. However, the high cost of fish meal has led to use alternative protein sources in catfish feeds.

Plant proteins can replace fish meal in commercial feeds but greater attention is required to satisfy dietary amino acid requirements (Wilson et al. 1981). One plant protein alternative is soybean meal. Lysine, one of the most limiting dietary essential amino acids in plant products, is also relatively abundant in soybean meal (Rumsey & Ketola 1975), but supplementation of soybean meal with crystalline lysine has been shown to enhance the growth and feed efficiency of channel catfish juveniles (Andrews & Page 1974). Bai and Gatlin (1994) concluded that the level and source of dietary protein significantly influenced the performance of channel catfish, and the benefits of supplemental lysine increased as the protein content of the diet declined.

2 Amino acids can be provided in the form of intact protein as feed ingredients or purified, -crystalline form as dietary supplements. The increased use of plant proteins in catfish diets during recent years led to increase use of purified amino acids to avoid deficiencies associated to high levels of plant products. For example, dietary L-lysine can be supplemented in purified form to eliminate the lysine deficiency in soybean meal (El-Saidy & Gaber 2003). Evidence for other fish than catfish (Cowey & Sargent 1979, Thebault et al. 1985) suggests that amino acids in intact protein are more efficient than those provided in crystalline form. This is, possibly due to the fact that crystalline amino acids are absorbed and catabolized more rapidly than amino acids from intact proteins.

Amino acid requirements can be met by feeding mixtures of complementary proteins and by supplementing deficient proteins with crystalline amino acids. The practice of using supplemental amino acids to improve the inferior quality of protein sources in catfish diets raises some questions. Studies have shown that crystalline amino acids may not be absorbed from the intestine in a manner that promotes effective utilization.

The best method to provide adequate amino acid nutrition to the channel catfish may be using a mixture of complementary proteins, in which different protein sources are mixed effectively to meet the dietary amino acid requirements. However, in some diet formulations, amino acids might have to be supplemented in purified form to meet minimum requirements.

1.2 Objective

The objective of this research is to study the effect of protein quality on determination of optimal daily protein supply for juvenile of patin (Pangasianodon hypophthalmus).

1.3 Hypothesis

Hypothesis of this research is: If protein quality differs then amino acid supply differs. Thus, the daily protein supply must be also different to meet amino acid requirement for optimal growth?

II.LITERATURE REVIEW

2.1 Protein and Amino Acid

Protein is a very important constituent of diet, both qualitatively and quantitatively, as it is the building material for the growing animal organism and is also important for the production of enzymes, among other things. Amino acids are the building blocks which formed proteins. There are 20 amino acids used as building blocks for the proteins in all living organisms. Amino acids composition in a feedstuff protein is of overriding importance for definition of its quality, and determines its value as a feed constituent (Steffens 1989).

Amino acids may be classified as either essential or non-essential. Ten amino acids are essential for fish, meaning that they cannot be synthesized in sufficient quantity for body needs; thus, they must be supplied in the diet. A non-essential amino acid is one that can be synthesized by the animal in sufficient quantity for maximal growth. Both essential and non-essential amino acids are required by body cells whenever proteins are being synthesized.

2.1.1 Fate of Protein

Utilization of dietary proteins in metabolism requires that the protein molecules were degraded to their constitutive amino acids. The reaction is catalyzed by a group of enzymes referred collectively as the proteolytic enzymes. The action of these enzymes is rather selective. The different enzymes preferentially split peptide bonds adjacent to particular amino acids. This selectivity depends upon the nature of the side chains of the amino acids.

Protein digestion takes place in the stomach and upper small intestine. When protein is digested, amino acids are released and absorbed into the body either as individual amino acids or as shorter chains of amino acids, di- and tri-peptides. It is rapid, and the products (the amino acid) are rapidly absorbed from the digestive tract. Dietary protein not only stimulates the secretion of proteolytic enzymes but it protects the enzymes against proteolysis, apparently because dietary protein denatured in the stomach is the preferred substrate for proteolytic enzymes.

4 Dietary protein

Undigested

Digested and absorbed

Feces Digestive enzymes

CO2 H2O NH3

Deaminated and used for energy Used to meet

protein needs

Urine

Dietary protein enters the portal bloodstream as free amino acid. These amino acids mix with those of endogenous origin to produce the amino acid patterns that are found in physiological fluids after ingestion of a protein-containing meal. Free amino acids within the body may follow three different metabolic pathways: (1) they can be used for protein synthesis; (2) they can serve as precursors in the synthesis of nitrogen-containing compounds such as nucleic acids, creatine, choline, or thyroxine; or (3) they can be degraded, the nitrogen being excreted mainly as ammonia and carbon skeleton entering energy metabolism.

Figure 1. The fate of dietary protein. Source: Lloyd et al. (1978)

Although proteins may serve as a source of energy, the primary reason for including protein in diet is to provide amino acids for the synthesis of essential tissues and fluid components, and to meet other specific needs for nitrogen.

5

2.1.2 Amino Acid Requirement

Cowey and Tacon (1983) suggested that the indispensable amino acid requirements of a fish should be related, or even governed, by the pattern of amino acids present in muscle tissue. Imbalances in the amino acid profile of the diet may depress feed intake and reduce efficiency of utilization of essential amino acid. Harper et al. (1964) suggested that plasma amino acids stimulate the synthesis or inhibit the breakdown of proteins in the liver which implies a decreased supply of the limiting essential amino acid in tissues. Consequently, the free amino acid pattern of plasma and tissue becomes imbalanced which is detected by the appetite regulating system in the brain. As a consequence, feed intake is reduced and this reduction on feed intake is at the origin of the growth depression effect of amino acid imbalances.

The ideal protein concept is based on the idea that there should be a direct correlation between the whole body amino acid pattern of the animal and the dietary amino acid requirements of the animal. This is essentially with the same A/E ratios (indispensable amino acid content/total indispensable amino acid content). The A/E ratios have been used as a means to estimate the requirements of all indispensable amino acids when only one is known by comparing the A/E ratio of indispensable amino acid to the A/E ratio of the known same amino acid (Wilson 2002).

Changes in the feed's amino acid composition should be made in a way that did not alter the relative ratios of amino acids. In other words, the ideal protein concept is built on the principle that amino acids are required and supplied in specific proportions to each other. Growing fish fed on such a diet would use a few amino acids for energy. The amino acids would be used efficiently for maintenance, health, and synthesis of proteins. This, will result in a maximum feed efficiency and growth.

Lysine is one of the ten essential amino acids which can be used as the amino acid reference. There are several reasons for selecting lysine as a referent amino acid. First, lysine major function in the animal body is protein tissue deposition; therefore, its requirement is not influenced by other metabolic roles. Second, depending on the fish species and type of ingredients, lysine is usually

6 the most or second most limiting amino acid; thus more is known about the requirements for lysine than other amino acids (Miles & Chapman 2007).

2.2 Patin (Pangasianodon hypophthalmus) 2.2.1 Classification and Morphology of Patin

Classification of patin according to Gustiano (2003) be as follows: Kingdom : Animalia

Phylum : Chordata Sub phylum : Vertebrata Class : Actinopterygii Sub class : Teleostei Order : Siluriformes Set of relatives : Pangasiidae Gender : Pangasianodon

Species : Pangasianodon hypophthalmus

Figure 2. Patin (Pangasianodon hypophthalmus). Source: www.fisheries.go.th

Pangasianodon hypophthalmus has a terminal mouth, with tooth band of upper jaw entirely covered by lower jaw when mouth is closed. Two pairs of narrow vomerine tooth band disappearing ontogenetically. Single chambered swimbladder with slender posterior part. This species is an air-breathing catfish that uses the swimbladder as an accessory respiratory organ (Brownman & Kramer 1985). The color of juveniles, typically with well defined midlateral and abdominal stripes separated at level of pectoral fin origin. Stripe although fainter posteriorly, sometimes extending to upper above end of anal fin. Larger specimens are less distinctively colored (Gustiano 2003).

Patin can live in a rather unfavourable environment. Generally, 5 mg L-1 dissolved oxygen concentration can support optimum fish growth. Dissolved oxygen concentration recommended for channel catfish reared in tank must be as

7 high as 3 mg L-1. Optimal water temperature for growth ranges from 28°-30°C, and pH 5-9 (Stickney 1993). Concentration of ammonia must remain less than 1.5 mg L-1 (Harimurti et al. 1995).

2.2.2 Protein and Amino Acid Requirement

Fish need feed with nutritional component which is appropriate to the size and the species. The basic diet for fish must contain sufficient levels of high quality protein and energy as well as balance essential fatty acids, vitamins and minerals. Information about nutrient requirement of patin remains scarce, thus information available for channel catfish are often used.

2.2.2.1 Protein Requirement

Young fish require higher protein level compared to larger fish as their metabolic and growth rate are higher (Stickney & Lovell 1977). Generally, catfish species requires protein level ranging 35-45% in its diet (NRC 1993). Gaylord and Gatlin (2001) reported that the growth rate of channel catfish increased when they fed on high protein (37%) and high energy diets (3.6 kcal DE/g). Protein requirements of channel catfish fry are higher than those of fingerling or adult fish. Protein levels of 52% and 40% are required for catfish fry and fingerling weighing 0.02 g and 20 g, respectively (Robinson et al. 1994).

Based on weight gain, FCR (Food Conversion Ratio), PER (Protein Efficiency Ratio) and ANPU (Apparent Net Protein Utilization11), Hung (1999) estimates the dietary protein requirement for Pangasius bocourti and P. hypophthalmus as 16 and 17 g CP kg-1 fish day-1, with an energy supply of 1150 and 900 kJ GE kg-1 fish day-1. For P. bocourti, P. djambal, and P. hypophthalmus, Hung et al. (2004) obtained 90% of the maximal specific growth rate for energy with a energy supply of 1212, 1084, and 952 kJ GE kg-1 day-1, respectively with a nearly 20 MJ per kg diet. In a study comparing utilization of carbohydrates in P. bocourti and P. hypophthalmus, growth rate was not affected by gross energy supply as high as 833 kJ GE kg-1 day-1 corresponding to a dietary energy content of 20 MJ kg-1 (Hung et al. 2003). For all these studies, feeding rates leading to

1

8 higher growth range from 30 to 50 g diet kg-1 day-1 with a dietary protein to energy (P/E) ratio from 16 to 20 mg CP kJ-1. Protein requirement for other catfishes is given in Table 1.

Table 1. Nutrient requirement of catfishes

Species

Requirement

Reference Protein

(g kg-1)

P/E Ratio (mg protein kJ-1 GE)

Fat (g kg-1)

Clarias anguillaris 400 Madu and Tsumba (1989)

C. batrachus 395 31* Mollah and Hussain

(1990)

310 24.2-25.8 Rabegnatar and Hidayat (1992)

C. gariepinus 400 Degani et al. (1989)

500 24* Henken et al. (1986) 430 20.5 82.3 Ali and Jauncey (2005)

C. isheriensis 370 22* Fagbenro (1992)

Heterobranchus bidorsalis

400 25* Kerdcheun (1992)

H. longifilis 425 26* Fagbenro et al. (1992)

Heteropneustes fossilis

280-350 Akand et al. (1989)

Ictalurus punctatus 320 25** NRC (1993)

**Satoh et al. (1989)

Mystus nemurus 420 27* Khan et al. (1993)

291 20,76 63.5 Kurnia (2002) 350 29.36 129.1 Suryanti et al. (2003)

Pangasius sutchi 250 Chuapoehuk and

Pothissong (1985)

P. djambal 677 32.55 Suhenda et al. (2004)

352 31.75 62.5 Suhenda et al. (2003)

P. hypophthalmus 354 28.24 40 Subamia et al. (2003) 296 26.46* 77 Syamsunarno (2008) * mg protein kJ/DE

2.2.2.2 Amino Acid Requirement

Amino acid requirements are influenced by interactions among the essential amino acids themselves, between essential and non-essential amino acids, and between amino acids and other nutrients. For example, if cystine is deficient in the diet, it can be synthesized by fish from methionine; the requirement for methionine is therefore partially dependent on the cystine content of the diet. A similar relationship also exists between phenylalanine and the non-essential amino acid tyrosine (Tacon & Cowey 1985).

9 Dispensable amino acids can be synthesized by catfish, but there are certain advantages if they are provided in the diet. Practical catfish feeds contain liberal amounts of dispensable amino acids inherent in the proteins of various feedstuffs. Channel catfish have been shown to require the same 10 indispensable amino acids as other fish. The quantitative requirements for these indispensable amino acids are summarized in Table 2.

Table 2. Amino acid requirement of catfishes Essential

Amino Acid

Requirement (g kg-1 protein)

African Catfish Channel Catfish Clarias hybrid

Arginine 45 43* 17.8-20

Histidine 15

Isoleucine 26

Leucine 35 35

Lysine 57 51

50*

48

Methionine +Cystine

32 23** 24

Phenylalanine +Tyrosine

50*

Threonine 20

Tryptophan 11 5

Valine 30

Reference Fagbenro et al. (1999)

Wilson et al. (1980) *Robinson et al. (1981) **Harding et al. (1977)

Unprasert (1994)

2.3 Amino Acid Supply

The dietary non-essential amino acids supplied in a diet, while not required, help to promote growth by reducing the need for amino acid synthesis, which saves energy (Tucker & Robinson 1991). During the digestion process, the rates of release and absorption of amino acids influence the dietary value of a protein source.

Proteins that contain an abundance of indispensable amino acids are commonly considered high-quality proteins. The radar plot is one of tools which can be used to evaluate the quality of protein. Figure 3 gives examples of radar plot for good and bad dietary protein content.

10 Figure 3. Dietary with good and bad protein content to satisfy catfish requirement.

From the radar plot above, supply from dietary with bad protein content can not satisfied catfish requirement; while the good protein can satisfied the catfish requirement, even some amino acids are in excess. Surplus amino acids supply by one protein has no adverse effect on the animal (Lloyd et al. 1978). This appears to be true for most diets composed with natural feedstuffs. If an indispensable amino acid is in excess then it will be catabolised, while if it is deficient the growth will be limited.

Crystalline amino acid supplementation is a way to override amino-acid deficiency resulting from incorporation of low quality protein in the diet. However, if growth improvements are observed the utilization of such crystalline amino acid can lead to various problems (Halver 1957).

Several researchers (Yamada et al. 1981, Murai et al. 1987, Schuhmacher et al. 1993) have demonstrated that amino acid concentrations in blood plasma increased more rapidly, and reached a peak level more quickly, after feeding crystalline amino acids than after feeding intact proteins. Cowey and Walton (1988), made similar observations when they measured the increase in radioactivity in the blood of rainbow trout after feeding pellets containing either

14

C-labeled amino acids or 14C-labeled protein. The lower growth rates and higher feed conversion ratios of rainbow trout fed crystalline amino acids have been attributed to a rapid uptake of amino acids from the gut into the peripheral blood,

0.0 5.0 10.0 15.0 20.0 25.0 30.0 Lys Arg His Ile Leu Met+Cys Phe+Tyr Thr Trp Val Catfish requirement Good protein Bad protein

11 leading to the deamination of the amino acids in excess and to higher rate of nitrogen excretion (Yamada et al. 1981, Cowey & Walton 1988).

Figure 4. Satisfying catfish requirement by increasing amount of protein bound-lysine.

Faster absorption of free amino acid may lead to transient amino acid imbalances and consequently to decreased protein utilization if crystalline amino acid is used to supplement dietary protein. Then giving dietary protein bound-lysine must be better than supplementation crystalline bound-lysine to meet requirement of amino acid. This can be achieved by mixing protein with complementary amino acid profile, or by increasing protein supply in order to provide adequate amount (on absolute basis) of the target missing amino acid. Considering the low quality protein example previously, a 59% increase in protein supply may meet catfish requirement for lysine (Figure 4).

0 5 10 15 20 25

Lys

Arg

His

Ile

0.5 Leu

Met+Cys 0.5 (Phe+Tyr)

Thr Trp

Val

Catfish requirement

Bad protein

Bad protein + 59%

Bad protein + crystalline lysine

III. MATERIALS AND METHODS

3.1 Rearing Condition

The experiment was conducted at Balai Riset Budidaya Ikan Hias (BRBIH) Depok, West Java with the support of Institut de Recherche pour le Développement. Fishes were 3-inch patin juvenile (Pangasianodon hypophthalmus) with an average mass 6.4±0.2 g. Density was 12 fishes per 60 L for each aquarium (50 cm x 40 cm x 40 cm). These fishes were obtained from Balai Besar Pengembangan Budidaya Air Tawar (BBPBAT) Sukabumi, West Java.

Before rearing, patin juvenile were being adapted to low fish meal diet during one week. The experimental period lasted 6 weeks with various daily protein supplies. Daily ration were given in the 3 equal meals at 08.00, 12.00 and 16.00. Aquariums were cleaned by siphoning every morning before first meal (07.00 am). Fish biomass was recorded every week to adjust daily ration in each aquarium. Prior to sampling, fishes were anesthetized with 600 µL L-1 eugenol solution (10% in ethanol).

Water quality (ammonia-N, pH, O2, temperature and conductivity) was

monitored weekly.

3.2 Diets

Feeds were prepared using fish meal (Chile), soybean meal (India), corn meal (local market), wheat gluten meal (Australia), palm oil (local grocery), corn oil, fish oil (Aphytec, France), vitamin, mineral (Agrobase®, France) and binder (Carboxyl Methyl Cellulose). The formulations were given in below (Table 3).

3.3 Chemical Analysis

Chemical analyses were conducted to measure proximate composition of feed ingredients, feeds, fish body and faeces. Proximate of feed ingredients was used as basis to formulate the diet and so proximate of diet after pelleting to check the composition. Proximate analysis of fish body was conducted at the beginning and

13 end of experimental period to measure protein and fat retention level. Proximate of faeces were conducted to measure total of feed digestibility.

Table 3. Diets formulation Ingredients Composition (g kg

-1

dry matter)

Diet HFM Diet LFM

Fish meal (76.4%) 131 24

Soybean meal (51.6%) 655 194

Corn meal (13.4%) 94 328

Wheat gluten meal (86.1%) 0 334

Palm oil 31 30

Corn oil 30 30

Fish oil 10 10

Vitamin 10 10

Mineral 30 30

Binder 10 10

Proximate composition (g kg-1dry matter)

Ash (%) 10.0 5.8

Crude protein (%) 46.8 46.3

Fat (%) 10.0 8.9

Crude fibre (%) 13.6 11.8

3.4 Feed Digestibility Test

Feed digestibility tests were conducted after treatment period. The diet was formulated to contain 5 g chromic oxide (Cr2O3) kg-1 in addition to experimental

formula. Fish were fed to satiation and faeces collected 2 hours after feeding with a siphon. Faeces were collected over one week after 2 days acclimation to the new diet (Moreau 1991). Samples were pooled before analysis and bigger fish were used to maximise the mass of collected faeces.

3.5 Experimental Method

Experimental design is a Complete Randomized Factorial Design (CRFD) with 2 factors, feed (2 levels) and protein supply (6 levels) using duplicates. Feeding rates were adjusted to meet protein supply according to protein in each diet. Experimental design is summarized as follow, with corresponding practical feeding rate for each treatment in brackets:

14 Table 4. Experimental design with fixed protein supply

1)

HFM = High Fish Meal (468 g CP kg-1), LFM = Low Fish Meal (463 g CP kg-1)

2)

FR (Feeding Rate) calculation using the formula:

FR = (Protein supply : Crude protein of diet) x 1000, ex. A1 = (8 : 468) x 1000 = 17.28 g kg-1 day-1

Linear model for this design is (Gasperz 1991):

Note:

Yij = Observation value for k experimental unit with ij’s treatment combination

(level i from factor A (diet) and level j from factor B (protein supply)) µ = Average of population

αi = Additive effect level i of A βj = Additive effect level j of B

(αβ)ij = Interaction effect between level i factor A and level j factor B Εijk = Error from k experimental unit within ij’s treatment combination

Effect of treatment can be tested using F test with 5% significance level. If effect is significant means compared using Tukey test with JMP® 7.0.2 program.

Diet1 Daily Protein Supply (g kg

-1

day-1)

8 14 20 23 26 30

HFM A1

(17.28)2 A2 (30.24) A3 (43.19) A4 (49.67) A5 (56.15) A6 (64.79)

LFM B1

(17.11) B2 (29.94) B3 (42.77) B4 (49.19) B5 (55.60) B6 (64.16)

15

3.6 Parameter

3.6.1 Specific Growth Rate

Growth rate was measured using SGR formula as follow (Guillaume et al. 2001):

Note:

SGR = Specific growth rate (% day-1)

Mf = Average fish mass at the end of experiment (g) Mi = Average fish mass at the beginning of experiment (g)

t = Observation period (day)

3.6.2 Survival Rate

Survival rate was measured as follow (Effendie 1997):

Note:

SR = Survival rate (%)

Nt = Number of live fish at the end of experiment (fish)

No = Number of live fish at the beginning of the experiment (fish)

3.6.3 Feed Conversion Ratio

Feed conversion was measured as follow (NRC 1977):

Note:

FCR = Feed conversion ratio (dry matter basis) F = Total feed intake (g)

Mf = Average mass at the end of experiment (g) Mi = Average mass at the beginning of experiment (g)

16

3.6.4 Protein Retention

Protein retention was measured as follow (Takeuchi 1988):

Note:

PR = Protein retention (%)

BPf = Average body protein at the end of experiment (g) BPi = Average body protein at the beginning of experiment (g) IP = Total protein intake per fish (g)

3.6.5 Fat Retention

Fat retention was value measured as follow (Takeuchi 1988):

Note:

FR = Fat retention (%)

BFf = Average body fat at the end of experiment (g) BFi = Average body fat at the beginning of experiment (g) IF = Total fat intake per fish (g)

3.6.6 Feed Digestibility

Apparent digestibility coefficient (ADC) was measured as follow (Takeuchi 1988):

IV. RESULT AND DISCUSSION

4.1 Result

Following 5 weeks of experiment, juvenile of patin fed with HFM and LFM diets survival was 100% and no deformity was reported. There was a trend of increasing mass gain with increasing protein supply in each diets, the lowest protein supply resulted in reduced mass gain (Figure 5 and 6).

Figure 5. Average mass gain (± SE) of patin fed on HFM diet during experiment according to protein supply in g kg-1 day-1. Each value corresponds to two observations.

5 7 9 11 13 15 17

0 5 10 15 20 25 30 35

A

v

e

ra

g

e

m

a

ss

(g

f

ish

-1)

Day

HFM diet

8

14

20

23

26

18 Figure 6. Average mass gain (± SE) of patin fed on LFM diet during experiment

according to protein supply in g kg-1 day-1. Each value corresponds to two observations.

Growth rates are shown in Table 4. Growth rate was significantly different between treatments. Higher levels of protein supply induced higher SGR values, and growth of fish fed on HFM diet was superior to fish fed on LFM diet with the same protein supply. The highest SGR (2.57%) was reached by using HFM diet with 23 and 30 g kg-1 day-1 protein supply. The HFM diet gave also the best FCR (0.9) with 8 g kg-1 day-1 protein supply.

5 7 9 11 13 15 17

0 5 10 15 20 25 30 35

A

v

e

ra

g

e

m

a

ss

(g

f

ish

-1)

Day

LFM diet

8

14

20

23

26

19 Table 5. Average initial and final body mass, growth rate, and food conversion

ratio according to diet and protein supply

Diet Protein supply (g kg fish-1 day-1)

Initial mass (g fish-1)

Final mass (g fish-1)

SGR

(% day-1) FCR

HFM 8 6.49 11.25abc 1.67bcd 0.90d

14 6.16 12.80abc 2.22ab 1.14cd

20 6.36 13.21abc 2.22ab 1.63bcd

23 6.56 15.40a 2.57a 1.58bcd

26 6.22 13.23abc 2.28ab 2.02b

30 6.33 14.81ab 2.57a 2.00b

LFM 8 6.66 10.02c 1.24d 1.23cd

14 6.26 10.31c 1.51bcd 1.76bc

20 6.41 12.60abc 2.04abc 1.85bc

23 6.44 9.96c 1.32cd 3.31a

26 6.43 10.92bc 1.60bcd 2.99a

30 6.52 11.73abc 1.78bcd 3.02a

SEM 0.15 0.74 0.14 0.13

Body compositions are shown in Table 5. Body dry matter, protein, ash, and gross energy (GE) did not differ significantly between treatments, whereas body fat content differed significantly. Highest body fat content (78) was observed with LFM diet and high protein supply, 23 g kg-1 day-1 while lowest fat content (42 and 46) was observed with HFM diet and 8 and 14 g kg-1 day-1 protein supply, respectively.

20 Table 6. Average of body composition according to diet and protein supply

Diet Protein supply (g kg fish-1 day-1)

Dry

matter Protein Fat Ash GE

Initial 205 138 31.4 31.3 4.50

HFM 8 212 137 41.9d 28.0 4.90

14 225 146 46.1d 29.1 5.27

20 225 143 48.7cd 27.5 5.29

23 232 147 51.7cd 27.3 5.52

26 248 158 50.1cd 34.2 5.71

30 222 134 56.7bcd 25.2 5.39

LFM 8 224 128 57.3bcd 31.0 5.28

14 225 127 62.2abcd 29.5 5.45

20 231 122 76.7ab 26.4 5.91

23 236 134 61.7abcd 30.7 5.59

26 245 130 78.0a 30.2 6.14

30 242 134 67.4abc 31.6 5.83

SEM 11 9 3.6 2.6 0.23

Nutrient utilizations are shown in Table 6. Protein retention, fat retention, and protein efficiency ratio (PER) differed significantly between treatments. Highest protein retention value (32.4%) was obtained with HFM diet and 8 g kg-1 day-1 protein supplies. Highest fat retention (100.2%) was obtained with LFM diet and 8 g kg-1 day-1 protein supplies. Highest PER value (2.37) was obtained with HFM diet and 8 g kg-1 day-1 protein supplies.

21 Table 7. Average of protein retention, fat retention, and protein efficiency ratio

according to diet and protein supply

Diet Protein supply (g kg fish-1 day-1)

Protein retention (%)

Fat

retention (%) PER

HFM 8 32.4a 62.0bcd 2.37a

14 29.2ab 52.8bcd 1.89ab

20 19.4bcd 39.8d 1.32cd

23 21.1abc 42.9cd 1.37bcd

26 18.7bcd 33.1d 1.06def

30 14.0cd 37.7d 1.07def

LFM 8 19.0bcd 100.2a 1.77bc

14 13.6cd 70.9abc 1.24de

20 12.4cd 76.2ab 1.18def

23 8.2d 39.9cd 0.65f

26 8.5d 54.7bcd 0.72ef

30 9.3cd 41.9cd 0.72ef

SEM 2.2 5.3 0.09

Dry matter and protein apparent digestibility coefficient (ADC) are shown in Table 7. Both dry matter and protein digestibility calculation gave similar values for the two diets, HFM and LFM. Nevertheless, LFM diet led to slightly better value than HFM with dry matter digestibility 89.0% and protein digestibility 94.4%.

Table 8. Average of feed digestibility

Diet ADC (%)

Total Protein

HFM 87.8 91.6

LFM 89.0 94.4

The amino acid profile of the two diets was compared with amino acid requirement of channel catfish (Figure 7). With HFM diet, the profile for each amino acid fulfilled amino acid requirement of channel catfish, even if excessive for most amino acids, while LFM diet appeared insufficient in lysine, but fulfilled for the others.

22 Figure 7. Amino-acid profile in each feed compared with channel catfish

requirement (g kg-1 protein).

4.2 Discussion

From the present experiment, the result showed that with the same protein supply, feed with higher fish meal content gave higher SGR than the lower fish meal one. SGR increased with protein supply for both diets. HFM diet gave the highest estimate for maximum SGR (2.57% day-1) which is reached when protein supply 23 g kg-1 day-1 and higher, while SGR still increase until 1.68% day-1 when protein supply reach 17.8 g kg-1 day-1 with LFM diet. The observed lower SGR values for both diets might be caused by too low protein supply, which can not cover protein synthesis for body mass allowing supporting optimum growth. Protein ingested by fish may presumably be catabolised to meet its requirement for maintenance rather than deposited for growth, when protein supply were too low.

Protein supplies required for maximum growth in the present study values are much lower than protein supply of 23.8 g kg-1 day-1 previously recommended for patin juveniles of the same size (Moreau et al. 2008). This may be related to the lower growth rate observed in the present study. Effectively, as fish may not display higher growth, protein deposition is lower and thus apparent protein requirement lower. Anyway, stricto senso protein requirement even for the same species may not be discarded from attended (or target) growth.

0 20 40 60 80 100

Lys

Arg

His

Ile

Leu

Met+Cys Phe+Tyr

Thr Trp

Val

Requirement

HFM

23 Figure 8. Broken line model adjusted to specific growth rate (SGR) of patin

juveniles according to daily protein supply with the two experimental diets over 33 days. Italic values indicated minimum protein supply for maximal growth with each diet.

FCR were generally improved with increasing protein supply, but HFM diet gave a better result than LFM when protein supplies are equivalent. Best FCR (0.90) are observed for HFM diet with 8 g kg-1 day-1. At this protein supply, feed consumption was restricted, nutrient input is better utilized by fish body even growth is limited. Differences in protein supply are associated to differences in feed intake which may act on feed efficiency. Higher result of FCR such as for most dietary LFM treatment indicated that utilization of this diet was less efficient. Furthermore, high FCR with higher protein supplies indicated that these protein supplies largely exceeded the optimal need of patin, and then food will be unavoidably wasted.

Body analysis indicated that dry matter, protein, ash, and GE gave the same results whatever the treatment. Fat increased with increasing protein supply on both diet. Carcass lipid levels were higher in fish fed dietary LFM. Highest fat composition (78) was achieved with LFM diet with 26 g kg-1 day -1, while the lowest (42 and 46) were obtained with HFM diet with 8 and 14 g kg-1 day -1 protein supply, respectively. LFM diet with 26 g kg-1 day -1 protein supply, a higher feed intake, led to highest value on fat composition indicating that lipid deposition was stimulated by high nutrient supply.

17.8 16.1 1.0 1.5 2.0 2.5 3.0

5 10 15 20 25 30 35

S G R (% d a y -1)

Protein supply (g kg-1_day-1₎

HFM LFM

24 Nutrient utilization, measured by protein retention, fat retention, and PER, decreased with increasing protein supply with both diets. With the same protein supply, HFM diet gave a better result for protein retention. Highest protein retention (32) obtained with HFM and protein supply of 8 g kg-1 day-1 indicated that rates of protein synthesis are higher than protein degradation and so provided more efficient retention of synthesized proteins. Highest fat retention (100) was achieved with LFM diet and protein supply of 8 g kg-1 day -1 might be caused by the fact that with this protein supply, fat content from LFM is deposited as storage lipid and/or neolipogenesis from COH.Lipid synthesis which occurred here needs energy and this will reduce energy available for growth. Experiments by Du et al. (2005) in carp juvenile and Mokoginta et al. (2004) in gouramy showed that higher fat content on diet, then lower fat which being deposited, on the contrary, lower fat content on diet, then higher fat which being deposited. Lowest fat retention (40, 33, and 38) was obtained with HFM diet at 20, 26, and 30 g kg-1 day-1, respectively corresponding to higher fat supply than 8 g kg-1 day-1.

The results for ADC showed that dietary LFM gave a slightly higher value for both dry matter and protein digestibility. Unfortunately, as experimental conditions did not allow replicates, the slight difference between the two feed was not tested. Overall, protein digestibility values (91.6 and 94.4%) were in a range (75–95%) reported for other freshwater fish fed practical selected diets (Kenan & Yasar 2005). In this experiment, diets that contained a high level of animal protein and those composed principally of plant-based ingredients were all highly digestible. This suggests a good efficiency of the pre-treatment process for these latter feedstuffs. This can be related to high efficiency of the thermal treatment, reducing trypsin and chymotrypsin inhibitory activities (Alonso et al. 2000). Then comparison between the two diets can be based on difference in amino acid supply rather than difference in protein availability.

In comparing the sum of differences between the total dietary levels of amino acids and the EAA pattern recommended by Wilson et al. (1980), it was apparent that the reference diet was closer to the requirement pattern, and that LFM diet had insufficient level of lysine. While in HFM diet, the profile was excessive for some amino acids. Several studies showed that excess amino acids

25 are catabolised for energy without interfering with the metabolism of other amino acids (Kaushik & Seilez 2010). The results showed that the provided dietary protein, regardless of source, was well digested by the fishes. Estimation of EAA content, such as lysine, is less in LFM than in HFM and also less than lysine requirement for channel catfish. Imbalances in the amino acid profile of LFM diet may depress feed intake and reduces efficient utilization of amino acid as excess in non-lysine amino acid may led to higher catabolism level.

Different in protein quality resulted on different amino acid supply and also optimal protein supply. HFM diet which fulfils amino acid requirement of patin’s body gave a better growth at higher protein supply than LFM diet. Moreover maximum growth was obtained with lower protein supply by HFM diet than by LFM. Thus, protein supply with LFM diet must be increased to provide sufficient amount of lysine in order to allow full utilization of all LFM amino acid. Then, it seems that higher protein supply may partially compensate unbalance amino-acid ratio. In the present study, higher protein supply is effectively required for maximum growth with LFM diet.

V. CONCLUSION AND RECOMMENDATION

5.1 Conclusion

1. Plant ingredients can partially substitute fishmeal in Pangasianodon hypophthalmus diets.

2. According to different protein quality, amino acid must be adjusted in order to reach optimal growth.

3. Increase in protein supply may partially compensate lower growth with lower protein quality.

5.2 Recommendation

Further experiments with graded variation of amino-acid ratio imbalance will allow a better understanding on the mechanisms of amino acid metabolism.

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Takeuchi T. 1988. Laboratory Work Chemical Evaluation of Dietary Nutrient, In: T. Watanabe (ed.): Fish Nutrition and Mariculture. Kanigawa Fisheries Training Center, Japan International Cooperation Agency, Tokyo. p 179-232.

Thebault H, Alliot E, Pastoureoud A. 1985. Quantitative methionine requirement of juvenile Sea-Bass (Dicentrarchus labrax). Aquaculture 50:75-87.

Tucker CS, Robinson EH. 1991. Channel Catfish Farming Handbook. New York: Van Nostrand Reinhold.

Unprasert NG. 1994. An evaluation of the use of “ideal protein concept to estimate essential amino acid requirement of the Clarias hybrid (Clarias macrocephalus x Clarias gariepinus). Ph.D Dissertation, Mississipi State University, Miss. State, MS.

Wilson RP, Poe WE, Robinson EH. 1980. Leucine, isoleucine, valine and histidine requirements of fingerling Channel Catfish. J. Nutr. 110:627-633.

Wilson RP, Robinson EH, Poe WE. 1981. Apparent and true availability of amino acid from common feed ingredients for Channel Catfish. Journal of Nutrition 111:923-929.

Wilson RP, Moreau Y. 1996. Nutrient requirements of catfishes (Siluroidei). Aquat. Living Resour. 9:103-111.

Wilson RP. 2002. Amino acid requirements of finfish and crustaceans. Departement of Biochemistry and Molecular Biology, Mississipi State University, USA. 52 p.

Yamada S, Simpson K, Tanaka Y, Katayama T. 1981. Plasma amino acid changes in Rainbow Trout force-fed casein and corresponding amino acid mixture. Bull. Jpn. Soc. Sci. Fish. 47:1035-1040.

34 Appendix 1. Proximate analysis of feed ingredients

Feed ingredients Proximate composition (g kg

-1

)

Dry matter Protein Fat Ash Fibre

Fish meal 911.5 763.8 84.5 148.4 -

Soybean meal 900.7 515.7 17.1 75.9 37.5

Corn meal 897.3 134.1 72.4 25.2 21.6

Wheat gluten meal 890.2 860.8 3.2 7.6 3.8

35 Appendix 2. Diet preparation procedure

Weigh each ingredients

Mix microingredient (mineral, vitamin, binder)

Mix macroingredient

(fish meal, soybean meal, corn meal, wheat gluten meal), spread evenly and then add microingredient mixture

while swirl until spread

Add fat (palm oil, corn oil, fish oil) slightly into those mixture

Add water to obtain a paste

Press with 1.5 mm dye

Dry pellet at room temperature

36 Appendix 3. Proximate analysis procedures

1. Dry matter analysis Material

Aluminium foil (shape as small dish) Analytical balance

Oven 105 °C Desiccators Tweezers

Procedure

Weigh aluminium foil (ME, mass empty), add ≈ 0.5 g sample (MF, mass fresh). Place aluminium foil containing sample at 105 °C for 12 hours (overnight).

Remove aluminium foil containing dried sample from oven and allow cooling in desiccators.

Weigh aluminium foil containing dried sample (MD, mass dry).

Calculation

37 2. Ash analysis

Material

Aluminium foil (shaped as small dish) Analytical balance

Oven 105 °C Furnace 500 °C Desiccator Tweezers

Procedure

Weigh aluminium foil (ME, mass empty), add ≈ 0.5 g sample.

Place aluminium foil with sample to 105 °C oven for 12 hours (overnight).

Remove aluminium foil containing dried sample from oven and allow cooling in desiccator.

Record mass of aluminium foil containing dried sample (MD, mass dry).

Ignite aluminium foil containing dried sample under fume hood. Transfer to furnace at 500 °C for 4 hours.

Remove aluminium foil containing burned sample from furnace and allow cooling in desiccator.

Weigh aluminium foil containing burned sample (MC, mass calcinated)

38 3. Crude protein analysis (Kjeldahl method)

Material

Crude protein apparatus with mineralizator, distillator, neutralizator and titrator

Analytical balance 1000 mL volume flask, 2 500 mL volume flask 1000 mL dark reagent flask 100 mL dark reagent flask, 2 1000 mL reagent flask 500 mL reagent flask 250 mL reagent flask 250 mL beaker glass 100 mL erlenmeyer

10 mL micropipette (pipetman) Aluminium foil

Magnetic stirrer Funnel glass, 3 3 mm glass marbles

Reagents

1. Sulfuric acid, H2SO4 96%

2. Sodium hydroxide, NaOH pellets 3. Ammonium sulphate, (NH4)2SO4

4. 96% ethanol, C2H5OH (EtOH)

5. Methylene blue 6. Methyl red 7. Boric acid, H3BO3

8. Kjeltabs (W) (7.5 parts sodium sulfate, Na2SO4; 1.5 parts copper (II)

39 9. NaOH, 40%

Weigh 400 g NaOH pellets (2) in a beaker glass 500 mL. Pour cautiously NaOH to jerrycan containing 1030 mL water . Mix until NaOH is completely dissolved in cold water. Store at room temperature.

10.Titration solution H2SO4 ( 1/28 M)

Weigh 3.65 g H2SO4 96% (1) in a beaker. Pour cautiously in a 1000 mL

volume flask containing approximately 500 mL of water. Mix. Adjust volume to 1000 mL. Transfer in 1000 mL reagent flask with titration system.

Store at room temperature. 11.Methylene blue 0.1 %

Weigh 0.05 g methylene blue (5) in dark reagent flask. Add 50 mL of EtOH (4). Mix.

Store at room temperature. 12.Methyl red 0.2%

Weigh 0.1 g methyl red (6) in dark reagent flask. Add 50 mL of EtOH (4). Mix.

Store at room temperature. 13.Indicator solution

Weigh 20 g boric acid (7) in a beaker glass. Pour cautiously in a 1000 mL volume flask containing 400 mL of water. Mix until boric acid is completely dissolved. Add 400 mL EtOH (4), 8 mL methylene blue 0.1% (11), 8 mL methyl red 0.2% (12). Mix. Adjust volume to 1000 mL. Transfer in dark reagent flask.

Store at room temperature. 14.Dilution indicator solution 1:1

Mixing 1 part colourant solution (13) for 1 part water . Store at room temperature.

15.Standard solution 1 mgN ml-1 (NH4)2SO4

Weigh 2.63 g (NH4)2SO4 (3) in a beaker glass. Pour cautiously in a 500

mL volume flask containing around 100 mL of water. Mix. Adjust volume to 500 mL. Transfer in labelled 500 mL reagent flask. Store at 4 °C.

40

Procedure

Weigh 0.1 g dry sample in a piece of aluminium foil (MS, sample mass) and transfer in Kjeldahl tube.

Mineralization

Add half of Kjeltabs® (8), 3 glass marbles and 10 mL H2SO4 96% (1).

Transfer to mineralization system with neutralization system, cover and heat on (temperature 375 °C) for 60 minutes.

Open the cover and remove Kjeldahl tube, then allow cooling.

Distillation

Heat up distillation apparatus. Repeat blank distillation until output reaches at least 80 mL.

Transfer Kjeldahl tube to distillation apparatus. Add 30 mL of water and NaOH 40% (9) until sample solution in tube turn to a brown or dark-blue solution.

Place down 100 mL erlenmeyer containing 10 mL indicator solution (14) and a magnetic stirrer in output system.

Start distillation during 5-6 minutes.

Do blank and standard using the same procedure distillation. For blank, use a clean tube and add 30 mL of water plus 20 mL NaOH 40% (9). For standard, add 5 mL (5 mg) standard (NH4)2SO4 (15) in a clean tube, then

add 30 mL of water plus 20 mL NaOH 40% (9).

Titration

Remove the 100 mL erlenmeyer containing ammonia extracted by distillation to titration apparatus.

Stir and titrate with 1/28 mol H2SO4 (10) until colour changes from green

to pinky-violet. Record volume of acid when colour changed (VA, sample volume). Do the same for blank (VB, blank volume) and standard (VS, standard volume).

41

Calculation

Standard measurement :

VST (mg N/mL acid) = 5 / (VS-VB) Crude protein concentration :

42

4. Fat analysis (Soxhlet Method)

Material

Soxhlet glass tube, with pump cooler Analytical balance

Oven @ 105 °C Desiccator

100 mL volumetric cylinder glass, 1 100 mL extraction baloon, 3

Glass funnel

Extraction thimbles, in glass container

Cotton (already washed with petroleum ether) Tweezers

Reagents

Petroleum ether (wasbensin)

Procedure

Weigh extraction ballon (ME, extraction flask mass), add 100 mL petroleum ether.

Weigh ≈ 1.0 gram dry sample in extraction thimbles with glass container (MS, sample mass), cover with cotton.

Transfer extraction thimbles into soxhlet glass tube. Connect extraction balloon to Soxhlet extractor. Turn on heater and allow boiling gently for 3 hours.

Turn off heater. Transfer extraction thimbles from Soxhlet tube to a glass container and pour petroleum ether from Soxhlet tube to a 2nd bottle for petroleum ether.

Turn on again until all solvent is trapped in Soxhlet tube. Pour petroleum ether in glass tube to the 2nd bottle.

Turn off, remove extraction balloon from heater and transfer to 105 °C for 12 hours (overnight).

43 Record the mass of extraction balloon containing fat extract (MF, mass extraction flask containing fat extract).

44

5. Crude fibre analysis

Material

Crude fibre apparatus, with 1000 mL beaker and reflux system Analytical balance

Filtration unit connected to a vacuum pump Oven @ 105 °C

Furnace @ 500 °C Desiccator

1000 mL beaker glass, 4 250 mL beaker glass, 2 250 mL cylinder, 1 250 mL reagent flask, 2 250 mL volume flask, 2

10 mL micropipette (pipetman)

Sinter glass crucible (volume 30 mL, porosity 1) Aluminium foil

Picker

Reagents

1. H2SO4, 40%

Weigh 104 g H2SO4 in a beaker. Pour cautiously in a 250 mL volume flask

containing around 100 mL of water. Mix. Cool under tap water flow. Adjust volume to 250 mL. Transfer in labelled 250 mL reagent flask.

Store at room temperature. 2. NaOH, 40%

Weigh 100 g NaOH pellets in a beaker. Add 200 mL water. Mix until NaOH is completely dissolved. Cool. Poor the solution cautiously in a 250 mL volume flask, adjust volume to 250 mL with water. Transfer in labelled 250 mL reagent flask.

Store at room temperature. 3. Antifoaming agent, 1-Octanol 4. Celite

45

Procedure

Weigh 0.5 to 1.0 g dry sample in a piece of aluminium foil (MS, sample mass).

Acidtreatment

Add 200 mL water in a 1000 mL beaker with 6.8 mL H2SO4 40% (final

concentration 1.25 %) and 1 g of Celite (4). Add 2 drops of antifoaming agent. Adjust reflux system. Heat up crude fibre apparatus (thermostat 3). Transfer sample into the beaker (thermostat 2). Adjust reflux system and allow boiling for 30 minutes.

Neutralization and alkalitreatment

Add 6.8 mL NaOH 40% to neutralize solution. This can be confirmed by the clarification of the solution.

Add further 6.8 mL NaOH 40% to adjusted final NaOH concentration to 1.25 %. Adjust reflux system and allow boiling for further 30 minutes (thermostat 2).

Add 6.8 mL H2SO4 40% to neutralize solution (thermostat 1).

Filtration

Adjust filtration device and pour supernatant on the filter. Repeat until all solution was filtered.

Wash filter twice with 30 mL hot water. For the last one, rinse the bottom of filter to remove any residue.

Drying

Remove filter from filtration device. Transfer to 105 °C oven for 12 hours (overnight).

46

Ignition

Remove crucible from oven and allow cooling in the desiccator.

Record mass of crucible containing acid-alkali extract (MD, mass of dry crucible containing acid-alkali extract).

Ignite crucible under fume hood. Transfer to furnace preheated @ 500 °C for 4 hours.

Remove crucible from furnace and allow cooling in the desiccator. Record mass of crucible with burned extract (MB, mass of crucible with burned acid-alkali extract).

Calculation

Crude fibre mass is given by the loss upon ignition corrected by the mass of filter:

47 Appendix 4. Random disposition of aquaria experiment

Water filter

Top Down Top Down Top Down

Note: A = HFM B = LFM

1 = protein supply 8 g kg-1 day-1 2 = protein supply 14 g kg-1 day-1 3 = protein supply 20 g kg-1 day-1 4 = protein supply 23 g kg-1 day-1 5 = protein supply 26 g kg-1 day-1 6 = protein supply 30 g kg-1 day-1

B4

A5

B2

A3

A4

A1

A4

B3

B6

A6

B2

A2

A2

B4

A3

A1 A5

B5

A6

B6

B1

B3

B5

Appendix 5. SGR and FCR calculation

Parameter

Treatment

HFM LFM

8 14 20 23 26 30 8 14 20 23 26 30

Initial average body mass (g)

1 6.72 6.23 6.30 6.68 6.02 6.07 6.67 6.14 6.40 6.67 6.29 6.59 2 6.27 6.09 6.42 6.43 6.42 6.60 6.65 6.37 6.41 6.20 6.57 6.45 Final average

body mass (g)

1 11.40 12.33 13.03 17.10 11.99 14.02 10.16 10.65 11.63 10.12 10.54 11.51 2 11.10 13.28 13.40 13.70 14.48 15.61 9.88 9.97 13.58 9.79 11.30 11.96 SGR (%) 1 1.60 2.07 2.20 2.85 2.09 2.54 1.27 1.67 1.81 1.26 1.56 1.69

2 1.73 2.36 2.23 2.29 2.47 2.61 1.20 1.36 2.27 1.38 1.64 1.87

Average 1.67±

0.09 2.22± 0.21 2.22± 0.02 2.57± 0.39 2.28± 0.27 2.57± 0.05 1.24± 0.05 1.51± 0.22 2.04± 0.33 1.32± 0.09 1.60± 0.06 1.78± 0.13 Total feed intake

(g)

1 4.42 7.58 11.09 14.67 12.83 15.92 4.11 7.14 10.65 11.98 13.49 15.63 2 4.16 7.46 11.19 12.71 15.27 17.96 4.10 6.98 11.92 11.30 13.31 15.76 FCR 1 0.94 1.24 1.65 1.41 2.15 2.00 1.18 1.59 2.04 3.47 3.17 3.17

2 0.86 1.04 1.60 1.75 1.89 1.99 1.27 1.94 1.66 3.15 2.81 2.86

Average 0.90±

0.06 1.14± 0.15 1.63± 0.03 1.58± 0.24 2.02± 0.18 2.00± 0.01 1.23± 0.07 1.76± 0.25 1.85± 0.26 3.31± 0.23 2.99± 0.25 3.02± 0.22

57 Appendix 14. ANOVA of dry matter

Source DF Sum of

Squares

Mean Square F Ratio

Model 11 2428,7138 220,792 0,9665

Error 12 2741,3665 228,447 Prob > F

C. Total 23 5170,0803 0,5191

Appendix 15. ANOVA of protein

Source DF Sum of

Squares

Mean Square F Ratio

Model 11 2363,9868 214,908 1,3507

Error 12 1909,3260 159,111 Prob > F

58 Appendix 16. ANOVA of fat

Source DF Sum of

Squares

Mean Square F Ratio

Model 11 2920,1560 265,469 10,0412

Error 12 317,2559 26,438 Prob > F

C. Total 23 3237,4119 0,0002

Level Least Sq

Mean

[LFM]26 A 77,963294 [LFM]20 A B 76,705626 [LFM]30 A B C 67,374393 [LFM]14 A B C D 62,200422 [LFM]23 A B C D 61,656346 [LFM]8 B C D 57,313974 [HFM]30 B C D 56,743536 [HFM]23 C D 51,680738 [HFM]26 C D 50,116073 [HFM]20 C D 48,707606 [HFM]14 D 46,072188 [HFM]8 D 41,860891

Levels not connected by same letter are significantly different.

Appendix 17. ANOVA of ash

Source DF Sum of

Squares

Mean Square F Ratio

Model 11 139,03060 12,6391 0,9151

Error 12 165,74012 13,8117 Prob > F

59 Appendix 18. ANOVA of GE

Source DF Sum of

Squares

Mean Square F Ratio

Model 11 2,5032428 0,227568 2,1515

Error 12 1,2692421 0,105770 Prob > F

60 Appendix 19. Amino acid calculation

Feed formulation (g kg-1 feed), feedstuff protein content (g kg-1 feedstuff), protein supply by each feedstuff (g kg-1 feed) and contribution of each feedstuff to dietary protein content (% dietary protein)

Feedstuffs Feed formulation Protein content

Protein supply Dietary protein content

HFM LFM HFM LFM HFM LFM

FM 131 24 764 100 18 22,2% 4,0%

SBM 655 194 516 338 100 75,0% 22,3%

WGM 0 334 861 0 288 0,0% 63,9%

CM 94 328 134 13 44 2,8% 9,8%

Total 450 450

Amino acid content from literature (g kg-1 as feed) and in each feedstuff (g kg-1 protein)

Amino acid

Feedstuffs (Guillaume et al. 2001)

Feedstuffs

FM SBM WGM CM FM SBM WGM CM

Protein 71.60 48.00 79.00 9.00

Lys 5.48 3.05 1.44 0.25 77 64 18 28

Arg 3.92 3.59 2.81 0.43 55 75 36 48

His 1.62 1.19 1.59 0.26 23 25 20 29

Ile 3.42 2.42 2.81 0.35 48 50 36 39

Leu 5.20 3.68 5.39 1.13 73 77 68 126

Met+Cys 2.80 1.43 2.65 0.39 39 30 34 43

Phe+Tyr 4.85 4.12 6.22 0.85 68 86 79 94

Thr 3.06 1.88 2.05 0.32 43 39 26 36

Trp 0.79 0.65 0.76 0.06 11 14 10 7

61 Amino acid profile for channel catfish requirement and in each feed (g kg-1 protein)

Amino acid Catfish requirement HFM LFM

Lys 50 68 33

Arg 43 72 48

His 15 25 23

Ile 26 51 41

Leu 35 80 78

Met+Cys 23 33 35

Phe+Tyr 48 85 84

Thr 21 41 31

Trp 5 13 11

62 Appendix 20. Water quality during experiment

Week Ammonia-N (mg/L) pH O2 (mg/L) Temperature (°C) Conductivity (µS)

1 87.86 4.98 6.26 28.5 349

2 54.06 5.74 5.77 27.6 294.3

3 74.09 5.81 4.31 29.9 322.7

4 53.52 6.1 5.46 29.5 304.2